cells

Review
Biomarkers in Hepatocellular Carcinoma: Diagnosis,
Prognosis and Treatment Response Assessment
Federico Piñero 1,2 , Melisa Dirchwolf 3 and Mário G. Pessôa 4, *
1 Hepatology and Liver Transplant Unit, Hospital Universitario Austral, School of Medicine,
Austral University, B1629AHJ Buenos Aires, Argentina; fpinerof@cas.austral.edu.ar
2 Latin American Liver Research Educational and Awareness Network (LALREAN),
B1629AHJ Buenos Aires, Argentina
3 Liver Unit, Hospital Privado de Rosario, 2000 Rosario, Santa Fe, Argentina; mdirchwolf@outlook.com
4 Division of Gastroenterology and Hepatology, University of São Paulo School of Medicine,
05403-000 São Paulo, Brazil
* Correspondence: mgpessoa@usp.br; Tel.: +55-11-2661-3340




Received: 5 May 2020; Accepted: 28 May 2020; Published: 1 June 2020


Abstract: Hepatocellular carcinoma (HCC) is one of the main cancer-related causes of death worldwide.
Thus, there is a constant search for improvement in screening, diagnosis, and treatment strategies to
improve the prognosis of this malignancy. The identification of useful biomarkers for surveillance
and early HCC diagnosis is still deficient, with available serum biomarkers showing low sensitivity
and heterogeneous specificity despite different cut-off points, even when assessed longitudinally,
or with a combination of serum biomarkers. In contrast, HCC biomarkers used for prognostic
(when associated with clinical outcomes) or predictive purposes (when associated with treatment
response) may have an increased clinical role in the near future. Furthermore, some serum biomarkers
are already implicated as a treatment selection tool, whether to provide access to certain therapies
or to assess clinical benefit after treatment. In the present review we will discuss the clinical
utility and foreseen future of HCC biomarkers implicated in surveillance, diagnosis, prognosis,
and post-treatment assessment.

Keywords: liver cancer; biological; markers

1. Introduction
Hepatocellular carcinoma (HCC) is nowadays one of the most frequent malignancies and a leading
cancer-related cause of death worldwide [1,2]. During the last decades, some improvements in the
therapeutic approach have been achieved not only for early but also for advanced HCC stages. On the
contrary, there has not been a significant clinical improvement in HCC biomarkers for surveillance and
early diagnosis. On the other hand, for prognosis and treatment response purposes, these biomarkers
might have a clinical role.
Although different serum or tissue biomarkers have already been studied, their clinical utility
has not been widely accepted. One of the most important issues, and maybe their Achilles heel,
is their low sensitivity (with high false-negative results), opposed to high specificity that precludes
HCC biomarkers to be clinically useful for early HCC diagnosis. Moreover, there is a huge amount of
publications with different and heterogeneous cut-offs with corresponding sensitivities and specificities.
However, most of these biomarkers have been associated with poor prognosis, either in early or
advanced HCC. Besides, tumor markers for appropriate treatment selection or response have been
widely evaluated in recent years. Even with the advent of new therapeutic oncologic modalities,
such as immunotherapy with checkpoint inhibitors [3–5], new biomarkers have not settled into daily

Cells 2020, 9, 1370; doi:10.3390/cells9061370 www.mdpi.com/journal/cells

Cells 2020, 9, 1370 2 of 27

practice, except for alpha-fetoprotein (AFP) [6]. Therefore, the challenge is to develop other biomarkers
for early diagnosis, adequate treatment selection of patients, and post-treatment prognosis, including
proteomics, metabolomics, genomics, and other novel biomarkers such as microbiome [7–10].
In this review, we describe HCC serum and tissue biomarkers focusing on their clinical utility
upon HCC surveillance, early diagnosis, prognosis, and post-treatment assessment.

2. Biomarkers for Hepatocellular Carcinoma Surveillance and Diagnosis

2.1. The Utility of Serum Biomarkers for Hepatocellular Carcinoma (HCC) Surveillance
Although the evidence-based data for HCC surveillance is low to moderate, including only
two randomized trials in patients with chronic hepatitis B infection (HBV) with a significant risk of
bias [11,12] and several observational studies in cirrhosis [13–16], surveillance for HCC is broadly
recommended by international guidelines [17]. This recommendation is supported by several
epidemiological reasons. First of all, HCC represents an important public health concern, currently
being the fourth cancer-related cause of death worldwide [2]. It has a well-defined population at risk,
including patients with chronic hepatitis B (HBV) or C virus (HCV), and patients with any chronic
liver disease with severe fibrosis or cirrhosis. These group of patients have an estimated cumulative
incidence of HCC above 1.5% per year [18]. Furthermore, due to its asymptomatic pre-clinical stage,
early diagnosis of HCC is feasible, providing higher access to curative treatments, and significant
improvement in overall survival [19].
It should be noted that the evidence regarding HCC surveillance is moderate in chronic HBV and
weak in patients with cirrhosis [11–16]. No randomized controlled trials assessing the survival benefit
of HCC surveillance in patients with cirrhosis were reported, and in some studies, factors such as
lead-time bias (apparent improvement in survival derived from early diagnosis) were not appropriately
considered [20]. However, due to the ethical conflicts of conducting a trial with a non-interventional
arm, current recommendations are based on the available evidence provided by cohort studies with
a significant risk of bias.
Finally, for a surveillance program to be successful, the screening test should be easily available,
cheap, reproducible, and with an appropriate detection accuracy [21]. Most scientific societies endorse
a 6-month interval evaluation with abdominal ultrasonography (US) [22]. The main drawback is the
fact that it relies on an operator-dependent method, thus offering heterogeneous results according
to the expertise of the imaging specialist [23]. Moreover, the potential benefits and harms of HCC
surveillance have been described with a 25% rate of false-positive results [24]. Asian countries have
proposed to include biomarkers other than AFP but in Western countries these were not included in
their recommendations [25–27]. Consequently, no international consensus has been reached so far
regarding the ideal biomarker to be used as a surveillance tool.

2.2. Difficulties Related to the Validation of Serum Biomarkers for HCC Surveillance

2.2.1. Alpha-Fetoprotein (AFP)

Alpha-fetoprotein (AFP) is the most commonly used biomarker for HCC surveillance. This 70 kD
glycoprotein is produced by the fetal liver and yolk sac during the first trimester of pregnancy and
declines rapidly after birth [28]. AFP is structurally very similar to albumin, with just a modification
in an N terminal sequence. It was first described as a useful biomarker for HCC over fifty years ago,
in murine models, and later in African and Siberian population studies [29]. Since its introduction as
a screening tool for HCC, its utility has been challenged.

The Specificity of Alpha-Fetoprotein

Serum AFP can be elevated in other benign or malignant conditions. Its specificity is undermined by
its elevation in other conditions such as acute and chronic hepatitis, intrahepatic cholangiocarcinoma,
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Cells 2020, 9, 1370 3 of 27

cholangiocarcinoma, and embryogenic tumors [29]. Particularly, elevated AFP levels in patients with
chronic HCV renders
and embryogenic tumorsthis [29].
biomarker conflictive
Particularly, for HCC
elevated AFPscreening
levels inpurposes in thischronic
patients with population
HCV
(associated
renders thiswith necro-inflammatory
biomarker conflictive foractivity). In the HALT-C
HCC screening purposestrial thatpopulation
in this included over 1000 HCV+
(associated with
patients with moderate to severe fibrosis [30], 11% and 27% of patients
necro-inflammatory activity). In the HALT-C trial that included over 1000 HCV+ patients with with bridging fibrosis or
cirrhosis
moderatehad AFP values
to severe above
fibrosis [30],2011%
ng/mL,
and respectively
27% of patients[31].with
Interestingly, only sixorpatients
bridging fibrosis cirrhosisdeveloped
had AFP
HCC,
valuesand only20three
above ng/mL,of them had elevated
respectively AFP values [31].
[31]. Interestingly, onlyBesides, at least
six patients 25% of the
developed HCC,patients had
and only
AFP values above 20 ng/mL at least once during follow-up without HCC development
three of them had elevated AFP values [31]. Besides, at least 25% of the patients had AFP values above [32]. More
recently,
20 ng/mLthe at lack
leastof reduction
once duringof AFP values
follow-up during
without HCCHCV treatment with
development [32].direct-acting antivirals
More recently, the lackhas
of
been proposed as an independent risk factor for HCC development; this
reduction of AFP values during HCV treatment with direct-acting antivirals has been proposed as approach is yet to be
validated [33]. Finally,
an independent it should
risk factor be clarified
for HCC that AFPthis
development; values have been
approach is yetreported in ng/mL
to be validated or IU/mL.
[33]. Finally,
Conversion
it should befrom IU tothat
clarified ng AFP
should be done
values havemultiplying
been reportedIU for 0.05 (e.g.,
in ng/mL 10 IU/mL
or IU/mL. is equivalent
Conversion fromtoIU
10.5
to
ng/mL).
ng should be done multiplying IU for 0.05 (e.g., 10 IU/mL is equivalent to 10.5 ng/mL).

2.2.1.2. AFP Cut-Offs

AFP Cut-Offs for Surveillance
for Surveillance and HCC andDiagnosis
HCC Diagnosis
The
The role
role of
of AFP
AFP asas aa screening
screening tool
tool and
and the
the selection
selection of of its
its most
most accurate
accuratecutoff
cutoff depends
depends on on the
the
prevalence
prevalence ofofHCC
HCCinin thethe tested
tested population.
population. Sensitivities
Sensitivities and specificities
and specificities of AFPvary
of AFP values values vary
according
according to which
to which cut-off cut-off
for HCC for HCC
early earlyisdiagnosis
diagnosis is selected
selected [34]. [34]. For
For a cutoff valuea cutoff value of
of 20 ng/mL, in20 ng/mL, in
a population
awith
population with a 5%of
a 5% prevalence prevalence of HCC,etTrevisani
HCC, Trevisani al. foundetaal. found apredictive
negative negative predictive
value (NPV)value of (NPV)
97.7%,
of 97.7%,
and and apredictive
a positive positive predictive
value (PPV)value (PPV)
of 25% of 25%
[34]. Thus, [34]. Thus, rendering
rendering this biomarker
this biomarker useful foruseful
exclusionfor
exclusion
of HCC but of HCC butits
poor for poor fordetection
early its early detection [34]. In comparison,
[34]. In comparison, when an when an HCC prevalence
HCC prevalence of 20% was of
20% was considered,
considered, this cutoffthishad
cutoff hadof
a PPV a PPV
61% of 61% maintaining
maintaining a NPVaof NPV
90%of[34].
90% Consequently,
[34]. Consequently, the
the AFP
AFP cut-off
cut-off with
with the the highest
highest sensitivity
sensitivity for earlyforHCC early
detection was of >20was
HCC detection ng/mLof >20
(60%)ng/mL
when (60%)
compared when to
compared to (>100
other cut-offs cut-offs>200
other ng/mL, (>100 ng/mL,
ng/mL >400 ng/mL
and >200 and >400
ng/mL) [34]. On theng/mL)
contrary,[34]. On the contrary,
specificities increased
specificities increased
with increasing with
cut-off increasing
values. Thus,cut-off values. Thus,
false negative and false
falsenegative
positive and
ratesfalse
varypositive rates vary
accordingly [34]
accordingly [34] (Figure
(Figure 1). These 1). Theseresults
heterogeneous heterogeneous resultswhen
were observed were AFP observed whenasAFP
was used was used
a screening as in
tool a
screening tool in different geographic areas, favoring its use in Eastern populations
different geographic areas, favoring its use in Eastern populations and poorly performing in Western and poorly
performing
populationsin[32,35].
Western populations
Racial [32,35].
disparities have Racial disparities
also been reportedhave [36].also been reported [36].

Figure 1. Sensitivities and specificities according to alpha-fetoprotein (AFP) cut-offs values for early
hepatocellular carcinoma (HCC) diagnosis. 1 Adapted from [34].
1 Adapted from [34].

Two meta-analyses evaluated the utility of adding AFP to abdominal US for HCC
Figure 1. [37,38].
surveillance Sensitivities
Theand specificities
first according13
study included to studies
alpha-fetoprotein (AFP) cut-offs
and described values for differences
no significant early
hepatocellular carcinoma (HCC) diagnosis.
in sensitivities for HCC detection at early stages with US alone (63%) and combined with AFP
(69%) [37]. In contrast, a recent meta-analysis including 32 studies found that US with vs. without AFP
Two meta-analyses
presented a significantly evaluated the utility
higher sensitivity of adding AFP
for early-stage HCCto(63%
abdominal
vs. 45%;USP=for HCC[38].
0.002) surveillance
It should
[37,38]. The first study included 13 studies and described no significant differences
be noted that the later meta-analysis observed a wide heterogeneity in ultrasound performance that in sensitivities for
HCC detection at early stages with US alone
could not be fully explained by subgroup analysis [38].(63%) and combined with AFP (69%) [37]. In contrast, a
recentThese
meta-analysis including
opposed results 32 studies
are reflected found
in current that
major US with
guidelines vs. without AFP
recommendations. Thepresented a
Asia-Pacific
significantly higher sensitivity for early-stage HCC (63% vs. 45%; P = 0.002) [38]. It should
Association for the Study of the Liver (APASL) guidelines endorse its use in combination with abdominal be noted
Cells 2020, 9, 1370 4 of 27

US [22], whereas the European Association for the Study of the Liver (EASL) guidelines state that
all tested biomarkers (including AFP) are suboptimal in terms of cost-effectiveness [25], and finally,
the American Association for the Study of Liver Diseases (AASLD) mention both surveillance strategies
(ultrasonography with or without AFP) as equivalent [26].

2.2.2. Suggested Approaches to Improve AFP Accuracy in Surveillance

Risk Stratification for Surveillance Algorithm

Another novel approach is to stratify the risk of HCC development, based on additional
scoring models. In HBV, the PAGE score has been developed in Caucasians and validated in
Asian population [39]. This score can identify patients with chronic HBV that are at higher risk of HCC
and require continuous HCC surveillance even after treatment with HBV antivirals. Among HCV
patients, other scoring models to assess the risk of HCC have been addressed including AFP values,
age, platelets count, and ALT levels [40,41].

Longitudinal Changes on Serum AFP

When the performance of dynamic changes of AFP was tested in a population with an HCC
prevalence of 3%, the combination of baseline AFP >10 ng/mL in combination with increasing AFP
levels increased the sensitivity to 80% with a NPV of 99% [42]. This encouraging approach is yet
to be validated. Another longitudinal approach based on Bayesian modeling was proposed based
on the HALT-C trial [43]. This modeling approach was proposed in patients with HCV considering
different cut-offs and longitudinal AFP changes during follow-up. It is still a matter of debate, whether
an AFP specific cut-off (>200 ng/mL) adds any clinical additional tool to detect HCC at an early stage
in patients with a negative US test.

Longitudinal Assessment of Combined Serum Biomarkers

Another recent novel approach is the sequential and longitudinal evaluation of combined HCC
biomarkers during a 12-month follow-up period [44]. In a case-control study nested on a prospective
observational cohort and in 3 randomized clinical trials of patients with chronic HBV, the longitudinal
assessment of AFP, DCP, and AFP-L3 were compared [44]. AFP had the highest AUROC for early
HCC diagnosis when compared to AFP-L3 (highly sensitive assay), and DCP. The combination of AFP
(cut-off >5 ng/mL) and AFP-L3 (cut-off >4%) showed the highest AUROC value (0.83) when compared
to any single biomarker [44]. However, while this resulted in an increased sensitivity, a decreased
specificity was observed with the combination of biomarkers [44].
Some authors have addressed the risk of developing HCC after HCV viral eradication with
clinical variables [45]. The presence of clinically significant portal hypertension is one of the most
important independent variables associated with the risk of developing HCC. Thus, among patients
with clinically significant portal hypertension, surveillance should be further stricter or underlined.
It remains uncertain whether the combination or longitudinal assessment of these biomarkers following
HCV eradication may optimize HCC detection rates at early stages, particularly in patients remaining
at higher risk of HCC.

2.2.3. Alpha-Fetoprotein Lens Culinaris Agglutin-3 (AFP-L3)

Total AFP can be separated into three fractions, AFP-L1 to AFP-L3, based on its reactivity to Lens
culinaris agglutinin (LCA) affinity on electrophoresis [28]. Of these three isoforms, the AFP-L3 fraction
appears to be more specific for HCC since it is produced exclusively by HCC cells. When initially
evaluated as a screening test, an elevated fraction (over 15%) of AFP-L3% showed slightly better
sensitivity than AFP (38% vs. 31%) for HCC detection [46]. It’s promising use as a surveillance test
was suggested from retrospective studies, in which 95% and 71% of the patients had positive values
of AFP-L3% at 3 and 6 months before diagnosis, respectively [46]. However, in further studies its
Cells 2020, 9, 1370 5 of 27

sensitivity and specificity greatly varied when cutoffs of 10–15% were used (from 36–96% and 89–94%
respectively) [47]. In a previously published metanalysis comparing AFP, AFP-L3 and Des-γ-carboxy
prothrombin (DCP) to detect a single HCC nodule of less than 5 cm, AUROCs were 0.65, 0.69, and 0.69
with corresponding cut-offs of >200 ng/mL, >15% and >40 mAU/mL, respectively [48]. When compared
with total AFP, AFP-L3% had a lower AUROC for differentiating cirrhosis from early HCC compared in
two case-control studies mainly enrolling non-Hispanic HCV patients [48]. However, in a prospective
cohort study from North America, a similar accuracy between AFP, AFP-L3%, and Des-γ-carboxy
prothrombin (DCP) was observed [49]. Only when these tests were used in combination, the sensitivity
increased to 77% while maintaining a high NPV (91%) for HCC surveillance [50]. Consequently,
it seems that the combination of these serum biomarkers might increase sensitivity at the expense of
decreasing specificity when compared to each biomarker alone [51].
Some technical issues must be addressed regarding the measurement of APF-L3 fraction.
In previously published papers, AFP-L3 was always measured simultaneously with AFP and its
significance depended on AFP values. Serum AFP values are usually measured by an immunometric
assay, whereas AFP-L3 levels by lectin-affinity electrophoresis coupled with antibody-affinity blotting.
It is expressed by the ratio of AFP-L3 to the total AFP in a percentage (%). However, AFP-L 3%
cannot be precisely measured in patients with low AFP values (<10 ng/mL) and in some patients
with higher AFP values (>400 ng/mL), the AFP-L3 proportion might not be accurately defined [51,52].
Consequently, in many previously published papers, the ratio and AFP-L3% has been artificially ranged
between 20 and 200 ng/mL of AFP total values. More recently, another technique was developed
through a micro total analysis system (micro-TAS), which has been proposed to be useful in cases
with AFP values below 20 ng/mL [53,54]. Thus, the greatest clinical utility of AFP-L3 or DCP has been
proposed to be in patients with intermediate AFP values (20–200 ng/mL); in which they have shown to
be highly specific for HCC [52].

2.2.4. Des-γ-Carboxy Prothrombin (DCP)

Also known as prothrombin induced by vitamin K absence-II (PIVKA-II), this abnormal protein
without coagulant function is presumably caused by an acquired defect in the posttranslational
carboxylation of the prothrombin precursor in malignant cells [55]. DCP has been described as a useful
tool for HCC surveillance since it is independent of AFP secretion. However, its efficacy as a screening
tool is still controversial. In a large-scale Chinese multicenter study evaluating the role of DCP in
HBV related HCC, DCP had better accuracy than AFP (88.5% vs. 76.2% respectively) as a surveillance
tool with a cutoff level of 40 mAU/mL [56]. Furthermore, the diagnostic accuracy of AFP plus DCP
was slightly improved when compared to DCP alone (1.12–2.69%) and significantly improved when
compared to AFP (8.26–13.42%) [56]. This biomarker also achieved a high accuracy in AFP-negative
HCC patients (AUROC of 0.86) [56]. In contrast, in a Caucasian HCV+ cohort, AFP was found to be
more sensitive than DCP for HCC diagnosis at early stages with a cutoff of 10.9 ng/mL [50]. Some
authors proposed that the combination of DCP with AFP and/or AFP-L3% may increase the sensitivity
and PPV for early HCC, whereas other authors described lower specificities when these biomarkers
were combined [55].
Some technical details should be addressed. First of all, DCP is not accurately quantified in the
presence of vitamin K deficiency, the use of oral anticoagulants, or in patients with a poor nutritional
status associated with alcohol abuse [57,58]. Moreover, in some cases, two different antibodies were
needed to accurately measure DCP (P-11 and P-16). The next generation method for DCP quantification
was created to avoid this latter technical issue [57,59].

2.3. Difficulties Related to the Use of Serum Biomarkers for HCC Diagnosis
The diagnosis of HCC has been settling down as a paradigm in clinical oncology. Through
its organ specificity, a non-invasive diagnosis can be done by a three-phase dynamic computed
tomography (CT) scan or a magnetic resonance image (MRI) in patients at high risk of HCC [25,26].
Cells 2020, 9, 1370 6 of 27

Specificity for HCC diagnosis is higher than 90% in these clinical scenarios, with the presence of specific
radiological hallmarks as arterial phase hyper-enhancement (APHE) and wash-out during late or
portal phases [60,61]. However, some nodules may not present these typical hallmarks or may present
with additional non-specific features. Thus, tumor biopsy may be appropriate in these cases [25,26].
In 2008, the American college of radiology proposed the Liver Imaging Reporting and Data
System (LI-RADS) to standardize the imaging interpretation and the reporting of nodular liver lesions
in patients at high risk of HCC [60,62]. The LI-RADS first version appeared online during 2011 and
since then it has been updated [62,63]. It categorizes the probability or likelihood of malignancy.
While LI-RADS 2 (LR-2) are more likely to be benign nodules, LI-RADS 3 to 5 have increasing
probabilities of HCC. LI-RADS M defines malignancy not specifically HCC [62,63].
The limited use of biomarkers for HCC diagnosis may be explained because during the last
decades, imaging technology has widely improved. Moreover, although the combination of serum
AFP, AFP-L3 and DCP have been proposed for HCC surveillance in Asia, their clinical use in Western
regions have not settled down. In this regard, the combination of these serum biomarkers added
a marginal improvement of HCC surveillance or diagnosis [52]. HCC biomarkers are elevated in a low
proportion of patients, they are associated with tumor burden and maybe not be useful for diagnosis at
early stages. Thus, low sensitivities and not enough specificities have been reported. Consequently,
imaging diagnosis is superior to biomarkers in daily practice. Nevertheless, in the context of a clinical
suspicion setting (population at risk) with atypical imaging features, the use of biomarkers may be
an additional tool for HCC diagnosis.
The poor performance of total AFP in detecting very early HCC has led to a past interest
in identifying tumor markers. In Japan, AFP-L3 and DCP in combination with AFP have been
proposed [64]. However, AFP-L3 and DCP have not been associated with a higher HCC detection
rate and both have been associated with larger tumors, metastatic disease, dedifferentiated tumors,
or vascular invasion [65,66]. Besides, elevated AFP-L3% are usually undetectable in patients with lower
AFP values (<10 ng/mL) and are usually seen in those with elevated total AFP [48,50,67]. As previously
mentioned, the combination of these biomarkers have been associated with increasing sensitivities but
decreasing specificities [68]. Consequently, most of the international Western clinical guidelines do
not recommend the diagnosis of HCC based on biomarkers [25,26]. In the end, the final aim of tumor
biomarkers should promote HCC diagnosis at early stages and a decrease in mortality rates [69,70]
(Table 1).

Table 1. Phases of biomarkers development and validation in cancer research.

Phase Design of Study Aims

I Preclinical
II Clinical—exploratory
III Observational—retrospective
IV Observational—prospective
V Trial—control
1Adapted from Early Detection Research Network (EDRN) of the National Cancer Institute from the United States
of America [69,70].
Identify clinical biomarkers
Detection of disease 1
Cancer detection at asymptomatic stages
Extent and characteristics of the disease
False referral rate
Impact on survival
Tumor progression

2.4. Scoring Models for HCC Surveillance and Early Diagnosis

Several authors have developed predictive models combining patient characteristics with serum
biomarkers for HCC surveillance and early detection. The GALAD score was developed including
Gender, Age, and a Logarithmic transformation of three biomarkers (AFP, AFP-L3, and DCP) [71].
This model showed a very good discrimination power with an AUROC of 0.97 irrespective of etiology
and disease stage. Other authors have proposed another scoring model for HCC surveillance,
the GALADUS score [71]. The authors evaluated the efficacy of the GALAD score in comparison to
Cells 2020, 9, 1370 7 of 27

abdominal US. The AUROC for the GALAD score (0.95; CI 0.93–0.97) was significantly higher than that
of US alone (0.82). This AUROC remained higher for the GALAD score irrespective of disease etiology
(US had a lower AUROC in patients with alcoholic liver disease), AFP values, or with the presence of
ascites in which the US performance is usually affected. Focusing on HCC detection at early stages,
the AUROC for GALAD score was still higher than US alone (0.92 vs. 0.82; P < 0.01). The combination
of GALAD and US presented even higher AUROC for HCC detection at early stages, particularly for
patients with a negative US result. Nevertheless, these results were based on retrospective cohorts
with some risk of bias. Although this approach is interesting, it needs to be prospectively validated.
The aim of these models should be to detect small HCC single lesions of less than 3 cm in diameter,
particularly in those cases with a negative US result.

2.5. Other Novel Serum Biomarkers for HCC Diagnosis

Other tumor biomarkers have been proposed such as osteopontin (OPN), vascular endothelial
growth factor (VEGF), angiopoietin 2 (ANG-2), Golgi protein 73 (Gp-73), insulin growth factor-1 (IGF-1),
hepatic growth factor (HGF), Glypican-3 and c-MET among others. Serum OPN has been associated
with increasing AFP serum levels, p53 mutation, vascular invasion, dedifferentiated HCC, and with
poor prognosis [72]. However, its accuracy in HCC detection at early tumor stages has not been
established. VEGF, AP-2, HGF and c-MET have been proposed as prognostic or predictive markers
and will be further detailed. Decreasing levels of IGF-1 in HCV+ cirrhotic patients were associated
with HCC development during follow up, independently from liver function [73]. This biomarker has
been proposed to identify patients with a preceding HCC diagnosis but has not been implemented in
daily practice. The Gp-73 has been assessed in a case-control study including HCC patients, cirrhotic
patients without HCC and healthy controls, matched for age, gender, and race [74]. The AUROC
outperformed AFP for HCC diagnosis (0.79 vs. 0.61) with a sensitivity and specificity of 69% and
75% for a cut-off value of 10 relative units. The AUROC for HCC detection at an early stage was 0.77
(95%; CI 0.70–0.85), with a sensitivity of 62%. Thus, a 38% false-negative rate precluded this biomarker
to be a novel tool for HCC surveillance. Serum dickkopf-1 (DKK1), a secretory antagonist of the Wnt
signaling pathway, was highly expressed in HCC tissue and not detectable in the non-tumor liver.
In a prospective HBV+ cohort, this serum biomarker was proposed to be a novel HCC biomarker with
a very good diagnostic performance for HCC, even in early stages and in patients with normal AFP
values (<20 ng/mL) (AUROC of 0.87 ;CI 0.83–0.91) [75].
Glypican-3, a cell-surface heparan sulfate glycoprotein, is highly expressed in HCC. In tumor
samples, it has been included for immunohistochemistry assays on pathology specimens. There is
no pathognomonic immunohistochemistry for HCC but the presence of at least 2 out of 3 markers,
Glypican 3 or Heat Shock Protein 70 or Glutamine sintetase, has 60% sensitivity and 100% specificity for
HCC [76]. Besides, Glypican-3 on tissue samples has been associated with poor prognosis in patients
with HCC [77,78]. It has been identified as a prognostic marker in two metanalysis. Liu et al. associated
Glypican-3 with aggressive histological tumor features, tumor progression, metastasis, and poorer
overall survival [78]. However, significant heterogeneity was reported when evaluating overall survival
(I2 of 65%) and disease-free survival (I2 of 81%) [78]. In another metanalysis, similar observations were
reported, associating Glypican-3 with aggressive histological tumor features (dedifferentiation and
vascular invasion). Higher expression of Glypican-3 on tissue samples was significantly associated
with worse overall and disease-free survival [77]. On the other hand, serum Glypican-3 has been
evaluated for surveillance and early HCC diagnosis [79]. Its NH2-terminal portion, which is soluble
(soluble GPC3), can be detected and quantified in serum samples. However, when compared to AFP,
Glypican-3 showed modest accuracy with a lower AUROC compared to AFP at a cut-off value of
20 ng/mL (0.72 vs. 0.80, respectively). Even when assessed in well-differentiated tumors, its AUROC
was not significantly better than AFP [79].
Genomic profiling and proteomics have gained popularity and are being focused on novel research
for HCC diagnosis during the last years [80,81]. Plasma micro-RNA has been linked to oncogenesis and
Cells 2020, 9, 1370 8 of 27

tumor metastasis. A large number of circulating micro-RNAs have been identified in HCC patients,
some of them associated with potential HCC diagnostic or prognostic implications [82–85]. However,
these novel biomarkers have been further tested as prognostic and predictive factors.

3. Serum Biomarkers in HCC for Tumor Staging and Prognosis

HCC is a unique tumor that includes not only tumor features but also remnant liver function as
key prognostic factors for survival. Thus, liver function, portal hypertension, and its complications act
as competing events for survival because most of the patients with HCC have underlying chronic liver
disease or cirrhosis [86–88]. According to different staging algorithms, biomarkers have or not been
included in these clinical staging systems.
It is important to specifically clarify the clinical implications and definition of prognostic and
predictive factors. Although these terms may sound similar, they are different. A prognostic factor is
an exposure baseline variable that it is independently associated with a worse clinical outcome. On the
contrary, a predictive factor may or may be not be a prognostic factor, but it identifies a population
with a better or worse response to a particular treatment.
The BCLC clinical algorithm has been proposed years before based on the most relevant scientific
evidence: it provides a rationale for the clinical-decision-making processes [89,90]. The BCLC includes
different prognostic clinical and tumor burden variables. Total bilirubin, presence of portal hypertension,
preserved liver function (absence of clinically significant portal hypertension, including ascites and its
complications) and Eastern Cooperative Oncology Group (ECOG) performance status are associated
with prognosis and were included in this algorithm.
However, biomarkers have not been included in the BCLC. Other staging proposals included AFP
values for HCC staging and prognosis, such as the Cancer of the Liver Italian Program (CLIP) [91],
the GRETCH staging from France [92] and the Chinese University Prognostic Index (CUPI) [93].
The Japan Integrated Scoring system integrates the TNM tumor staging with the Child–Pugh
score [94,95]. More recently, another staging system proposed by the Hong Kong Liver Cancer
group did not include AFP values or other biomarkers to the clinical-decision-making processes [96]
(Table 2).

Table 2. Clinical staging algorithms for hepatocellular carcinoma (HCC) and inclusion of biomarkers.

Hepatocellular
Clinical Variables
Staging Algorithm Tumor Variables Carcinoma (HCC)
Included
Biomarkers
Number and diameters
ECOG
nodules
BCLC [89,90] Preserved liver function Not included
Vascular invasion
Portal hypertension
Extrahepatic spread
Number and diameters
ECOG nodules
HKLC [96] Not included
Child–Pugh Vascular invasion
Extrahepatic spread
JIS [94,95] Child–Pugh TNM Not included
Tumor extension >50%
CLIP [91] Child–Pugh AFP > 400 ng/mL
of liver volume
Karnofsky index
GRETCH [92] Total bilirubin Portal thrombosis AFP > 35 ng/mL
Alkaline phosphatase
Asymptomatic
Ascites
CUPI [93] TNM staging AFP > 500 ng/mL
Total bilirubin
Alkaline phosphatase

Other authors proposed to assessed HCC staging excluding imaging or clinical data [97,98].
These new models based on biochemical data with or without biomarkers, have been developed
Cells 2020, 9, 1370 9 of 27

to surpass the subjectivity of scores and grading systems based on clinical data to assess liver
function (Child–Pugh score, including ascites or encephalopathy grades). The BALAD score includes
2 biochemical variables (serum bilirubin, albumin) and three biomarkers [97]. This scoring model
included a panel of 3 biomarkers (AFP >400 ng/mL, AFP-L3 >15% and DCP >100 mAU/mL) and
their combination was associated with worse prognosis [64]. However, as this score does not include
any imaging tumor features or clinically relevant data, it has not been widely implemented in daily
practice. Moreover, although survival was well assessed, there might have been a treatment selection
bias. The discrimination power of BALAD and BALAD-2 scores were not superior to other clinical
staging algorithms that did not include biomarkers, as the JIS [99,100]. Nevertheless, the BALAD
score was recently externally validated in Asian and Western populations [99–101]. Although it was
designed for HCC detection rather than prognosis, the BALAD-2 score was found to be associated
with prognosis in another external validation cohort [102]. More recently, the GALAD score has been
validated in patients with non-alcoholic fatty liver, either with or without cirrhosis [102].
The ALBI grade was developed to avoid any subjective bias interpretation of clinical data,
including ascites or other portal hypertension complications [98]. It did not include performance status.
Although novel and interesting, this score presented a modest discrimination power with a Harrells’
c-statistic lower than 0.70 in the test and validation cohorts. It did not add any significant gain on
discrimination power over the Child–Pugh score [98] (Table 3).

Table 3. Staging models for HCC prognosis including biomarkers.

Tumor Imaging Biochemical or HCC

Staging Algorithm Clinical Variables
Features Biomarkers
serum bilirubin, albumin,
BALAD score AFP > 400 ng/mL,
None None
[99,100] AFP-L3 > 15% and DCP
> 100 mAU/mL
ALBI grade [98] None None serum bilirubin, albumin

3.1. Biomarkers as Clinical Prognostic Tools

3.1.1. Alpha-Fetoprotein
AFP has been widely and extensively studied in association with prognosis. However, controversy
has been raised regarding which specific cut-off for survival or recurrence should be chosen.
Several cut-offs have been proposed associated with worse survival. Nevertheless, increasing AFP
values are associated with lower survival and higher tumor recurrence rate in patients at very early or
early stages [103–106], as well as poor prognosis in patients with advanced HCC [107].
In patients at very or early stages, AFP values above 1000 ng/mL were associated with worse
survival and higher recurrence rates [108]. On the other hand, this biomarker has been correlated
with microvascular invasion and tumor dedifferentiation on explant pathology analysis after liver
transplantation (LT) [103,105]. Consequently, AFP values are useful for selection of appropriate or
best candidates for surgical options or LT. In this regard, since 2013, France has adopted the AFP
model, which includes AFP values and radiological tumor burden, as a selection tool for transplant
candidates [103]. Besides, in the United States of America, a restriction policy for LT has been
implemented with AFP serum levels above 1000 ng/mL [109]. Other authors have proposed other
cut-offs [110,111] or continuous AFP values [106].
In patients at intermediate stages, AFP has been associated with tumor progression in patients
listed for LT who underwent tumor bridging therapies while on the waiting list or in patients who
received locoregional tumor treatment to reduce their tumor burden. This latter objective, called
downstaging from beyond to within tumor transplant limits [112,113]. Serum AFP values above
400 ng/mL have been associated with higher waitlist tumor progression and a lower response rate
Cells 2020, 9, 1370 10 of 27

after trans-arterial chemoembolization (TACE) [114,115]. Moreover, AFP values above 100 ng/mL have
been recently proposed as a selection criterion for the best candidates for downstaging [116].
Among patients with advanced HCC, serum AFP values have been associated with worse baseline
prognosis in the SHARP and Asia Pacific trials evaluating the efficacy of sorafenib [117,118]. Based on
these trials, AFP values above 200 ng/mL were associated with a poor prognosis in both treatment
arms; however, sorafenib was effective even in this population [107]. Although AFP was associated
with lower survival in this latter group, it was not included as a stratification factor in some trials
assessing first line systemic treatment options [119–123]. AFP values have been associated with worse
clinical outcomes in patients with tumor progression during sorafenib treatment [124]. Consequently,
other trials for second line systemic treatments have included it as a stratification factor [125] or as
an exclusion eligibility criteria [5]. Moreover, AFP values above 400 ng/mL have been associated with
response criteria for ramucirumab; thus, showing to be the first biomarker used as selection criteria for
systemic treatment options [6].

3.1.2. Alpha-Fetoprotein LP-3

AFP-L3 has been widely and extensively studied. This biomarker has been associated with
biologically more aggressive tumors too. Originally described to be associated with multiple HCC
recurrences, vascular tumor invasion, dedifferentiated tumors, and poorer overall survival [65,126,127].
Elevated AFP-L3 levels have been associated with a higher risk of HCC recurrence and early recurrences
following tumor resection [128]. In other observational studies, the combination of AFP, AFP-L3,
and DCP were associated with lower overall survival, larger tumors, and vascular invasion [97].
The prevalence of each tumor marker in that study showed that 23.2% of the cohort did not have
any positive biomarker (cut-offs were for AFP >20 ng/mL, AFP-L3 >10% and DCP >40 mAU/mL),
32.1% had at least one positive biomarker, 22.3% 2 out of 3 and 22.3% 3 positive biomarkers [97].
In another cohort study evaluating the prognostic effect of combined biomarkers following liver
resection, the recurrence-free survival (RFS) at 2-years of follow up was significantly lower with
the presence of 1, 2 or 3 biomarkers including AFP, AFP-L3 and DCP (55% vs. 38% vs. 19%) [129].
There was an increasing prevalence of microvascular invasion or poorly differentiated tumors [129].
An elevated fraction of AFP-L3 has been linked with poor prognosis in surgically resected patients
with HCC in Japan; associated with poorly differentiated tumors but showing less specificity for
vascular invasion when compared to DCP [66]. The highly-sensitive AFP-L3 with a 5% cut-off value
was associated with lower overall survival, even in patients with AFP values less than 20 ng/mL [54],
and a higher risk of HCC recurrence after hepatectomy [130,131].
Finally, in a North American cohort study, the highly sensitive AFP-L3 in combination with AFP
and DCP serum levels were associated with a higher risk of HCC recurrence after LT [132]. Indeed,
the authors proposed a novel selection tool for patients beyond Milan criteria [133] using different
cutoffs of AFP (>250 ng/mL), AFP-L3 (>35%) and DCP (>7.5 ng/mL) [132]. The inclusion of each
biomarker with Milan criteria as a novel selection criterion showed that corresponding AUROCs were
for Milan + AFP 0.68 (CI 0.60–0.76), AFP-L3 0.70 (CI 0.62–0.78) and DCP 0.70 (CI 0.62–0.78). However,
a significant selection bias was observed due to the exclusive inclusion of HCC patients without HCC
recurrence after LT; those without recurrence were discarded for blood samples storage and analysis.
Thus, the association between these biomarkers and post LT HCC recurrence was biased in this study.

3.1.3. DCP
DCP or PIVCA-II has also been explored as a prognostic marker in HCC. It has been widely
associated with larger tumors, poor differentiation, and vascular invasion [127,134]. It has also been
shown that its serum levels are higher after tumor hypoxia, and has been proposed as a predictive
biomarker after anti-angiogenic therapies [135]. However, its prognostic value has been evaluated
with different cut-off values [127]. The most commonly used was >40 mAU/mL either alone [127] or in
Cells 2020, 9, 1370 11 of 27

combination with other biomarkers [129,136]. It was specifically associated with vascular invasion
compared to AFP or AFP-L3 [68].
In regard with clinical outcomes, DCP has been associated with lower survival and a higher
risk of HCC recurrence following liver resection, with higher specificity (92% vs. 87%; P < 0.001)
and sensitivity (74% vs. 41%) compared to AFP values [137,138]. On the contrary, it has been
associated with a lower discrimination power for HCC recurrence after living donor LT (LDLT) in
Japan [139]. The AUROC for AFP, DCP and the neutrophil/lymphocyte ratio were 0.88, 0.76 and
0.62, respectively [139]. In this study, the authors proposed a new selection criteria for LDLT with
a scoring model including the number of nodules (less or equal than 5), largest tumor diameter less or
equal than 50 mm and the presence/absence of AFP >250 ng/mL, DCP >450 mAU/mL (LDLT Tokio
University criteria) [139]. With the presence of at least 2 or 3 variables, the disease-free survival (DFS)
and overall survival at 5-years following LT were 20% and 20%, respectively. Similarly, other authors
proposed the Kyoto LDLT criteria incorporating DCP >400 mAU/mL, independently associated with
HCC recurrence after LT [58]. The new Kyoto criteria included tumor number less or equal than 10,
the largest nodule diameter less or equal than 50 mm, and DCP >450 mAU/mL. The AUROC was 0.84
compared with AFP [58]. Other authors have addressed lower cut-offs for HCC recurrence following
LT (<300 mAU/mL) [140] or microvascular invasion in early HCC (>90 mAU/mL) [141] (Table 4).

Table 4. Biomarkers used for liver transplantation selection criteria in patients with HCC.

LT Criteria Tumor Imaging Features Biomarkers Expected Outcomes

Total tumor volume (TTV)
AFP tumor volume [110]
>115 cm3
Tumor number (1–3 vs.
≥4 nodules)
The AFP model [103]
Largest diameter (≤3 cm,
3–6 cm and >6 cm)
Hanghzou criteria [111] Sum of diameters (≤8 cm)
Sum of nodules and largest
Metroticket 2.0 [106]
diameter (Up-to-7)
Tumor number Beyond Tokio criteria
Tokio criteria [139] (≤5 nodules)
Largest diameter (≤5 cm) DCP > 450 mAU/mL
Tumor number
Kyoto criteria [58] (≤10 nodules)
Largest diameter (≤5 cm)
3-model biomarker
Beyond Milan AFP-L3 (>35%) or DCP
approach [132]
AFP >400 ng/mL Overall survival <50% at 3 years
AFP ≤ 100 ng/mL, AFP score 2-point cut-off:
101–1000 ng/mL and 5-year survival and recurrence
>1000 ng/mL of 67.8% and 8.8%
Within Hanghzou:
AFP > 400 ng/mL 5-year survival and recurrence:
70.8% and 35.7%.
Three different thresholds for
Log10 AFP HCC specific survival rate >50%
at 5 years.
2/3 criteria:
AFP > 250 ng/mL
5-year survival 20%
Beyond Milan & within Kyoto
DCP > 400 mAU/mL
criteria: 5-year recurrence 4%
AFP (>250 ng/mL) or
Higher recurrence with any of
these criteria.
(>7.5 ng/mL)

3.1.4. Osteopontin
This tumor biomarker has been associated with pathological features of aggressive HCC, including
dedifferentiation and vascular invasion [72].

3.1.5. Other Novel Biomarkers

Another promising biomarker associated with worse prognosis in early and advanced HCC
stages was the HGF, known to promote tumor growth and metastasis. Encoded by the MET gene,
HGF tyrosine kinase receptor inhibition was proposed as a potential therapeutic target [142]. The MET
proto-oncogene encodes the tyrosine kinase receptor for the HGF, which activates downstream
mechanisms upon tumor proliferation, invasion, and anti-apoptotic signals. MET overexpression in
tumor tissue samples was associated with worse prognosis in patients receiving sorafenib. Indeed,
Cells 2020, 9, 1370 12 of 27

expression of HGF/MET was observed to be increased after sorafenib exposure, suggesting an oncogenic
escape mechanism [143]. However, a trial evaluating the effect of tivantinib, a MET pathway inhibitor,
in patients highly expressing MET following sorafenib exposure, did not show any survival benefit
compared to placebo [144]. Another MET inhibitor has recently shown efficacy for second-line
treatment in advanced HCC [145]. Other angiogenic pathways have been associated with poor
prognosis, including VEGF and ANG-2, among others, associated with vascular invasion, advanced
tumor stages, lower DFS, and overall survival [146]. Micro-RNAs have also been linked to worst clinical
outcomes in different observational studies [147] and are now being analyzed in large gene expression
databases [148]. The neutrophil/leucocyte ratio (NLR) has been associated with worst overall survival
in patients treated with sorafenib for advanced HCC [107] but it has not clearly shown to be associated
with clinical outcomes in other clinical settings [139], although linked to HCC recurrence in other
studies [149].

4. Serum and Tissue Biomarkers in HCC for Response Assessment Following Tumor Treatment
Clinical research has focused on the predictive capacity of each biomarker to evaluate tumor
response before or after specific treatments. Apart from its prognostic association already described
and their potential role in candidate selection, some biomarkers have been associated with a better
response after treatment for early, intermediate, or advanced HCC. However, only AFP has been
assessed as a selection tool for better candidates for liver transplantation [103,105,106,150] or a specific
anti-angiogenic therapy [6].
One of the main proposed explanations is that HCC is a very heterogeneous tumor from a genetic
standpoint with different oncogenic pathways [80]. The other hypothesis is that a higher expression of
specific oncogenic pathways may not correlate with better clinical outcomes or treatment response.
Moreover, although some biomarkers have been established as predictive factors for specific treatment
in other cancers, in HCC this has not been the case. Immunotherapy based on checkpoint inhibitors
in HCC has not been associated with increasing or decreasing efficacy based on PD-1 or PD-L1
assessment [3,5,151]. Previously, several directed-molecular therapies have failed to show any survival
treatment benefit. One example was the high expression of c-MET on tumor tissue that showed
promising results in a phase II trial [152], but did not show a survival benefit in second-line systemic
treatment for advanced HCC [144].

4.1. Predictive Serum Biomarkers Following Locoregional Treatment

Decreasing levels of serum biomarkers might serve as a potential predictive marker of better
outcomes. Indeed, AFP-L3 dynamic changes before or after locoregional HCC therapy or surgical
resection has been associated with better overall survival in some observational studies [126,153].
PIVKA or DCP has also been associated with better outcomes as prognostic factor but it was not
identified as a predictive factor of treatment response assessment. Indeed, DCP serum level at
cut-off of less than 40 mAU/mL has been associated with better survival and lower recurrence rate in
an HBV cohort after radiofrequency ablation [154]. VEGF serum level is a baseline prognostic factor
following trans-arterial chemoembolization (TACE) [146]. This observation led to design a phase III
trial evaluating the combination of TACE with sorafenib [155,156]. However, this biomarker did not
correlate with better treatment response.

4.2. Predictive Serum Biomarkers Following Systemic Treatment

In the last years, systemic therapy for advanced HCC has improved the prognosis due to
newly available drugs for first- and second-line systemic treatment options. During the last decades,
further knowledge of HCC molecular mechanisms has led to the development of effective systemic
treatment including tyrosine kinase inhibitors (TKIs) and immunotherapy. Sorafenib, the first
antiangiogenic agent to be approved for first-line systemic treatment for advanced HCC, has shown
to be effective irrespectively from disease etiology and baseline tumor biomarkers, including AFP,
Cells 2020, 9, 1370 13 of 27

VEGF or ANG-2 [157,158]. However, no identified biomarker has been able to select appropriately
the best candidates for any specific therapy; except from AFP serum levels and ramucirumab in
second-line systemic therapy for advanced HCC [6]. Serum AFP, VEGF, and ANG-2 baseline levels
were shown to be prognostic factors but were not predictive factors for sorafenib efficacy [107,159].
High baseline c-KIT and low baseline HGF were predictive factors of sorafenib treatment efficacy
on univariate analysis and had a trend to be independently associated after adjustment for other
prognostic factors [159].
A novel clinical term coined 10 years ago but yet not implemented in the daily practice, is the
so-called “AFP response” [160]. This concept is the decreasing levels of AFP associated with better
outcomes, but it was not identified as a predictive marker of better response [161]. A decreased in more
than 50% from baseline levels of AFP following trans-arterial chemoembolization or trans-arterial
radioembolization, defined as “AFP responders“ has been associated with better overall survival
(HR 2.7 (CI 1.6–4.6) for mortality); however, the effect was not independent of that observed and
evaluated through radiological imaging criteria [161,162]. The novel approach for “AFP response”
might have a clinical role in patients under TKI, in which tumor shrinkage or a partial or complete
radiological response is an infrequent event [117,118]. Another clinical definition of “AFP response”
after systemic treatment might be a 20% decrease from baseline during the first 2–4 weeks of treatment
initiation [160,163] or more than 50% at the end of the first month [164]. On the contrary, DCP has
also been assessed following sorafenib, and a 2-fold increase in its serum levels might be a predictive
factor of better response [165]. This preliminary observation was not further validated. However,
there might have been several selection biases when assessing the “AFP response” as a prognostic
variable. In some of these retrospective cohort studies, AFP baseline values in the group of “AFP
responders” were significantly lower when compared to non-responders. The appropriate comparison
should have been made in patients with baseline AFP levels >200 ng/mL [107,157].
Other predictive factors have been tested more recently in first and second-line systemic treatment
for advanced HCC. Lenvatinib has been approved as a first-line systemic treatment option to sorafenib
in an open-labeled phase 3 randomized clinical trial (REFLECT) [123]. In this trial, baseline VEGF,
ANG-2, and fibroblast growth factor-2 (FGF-2) serum levels were associated with better overall survival
with lenvatinib but not with sorafenib [166]. Besides, increasing VEGF levels following lenvatinib
initiation was associated with better overall survival [166]. Tumor markers response and assessment
through imaging data were evaluated in another retrospective cohort study including pre and post AFP
or DCP levels at weeks 2 and 4 from lenvatinib initiation and correlated with the modified Response
Evaluation Criteria for Solid Tumors (mRECIST) criteria [167,168]. In second-line systemic therapy,
data coming from the RESORCE trial of regorafenib versus placebo including plasma and tissue
samples showed that baseline serum levels of AFP and c-MET were associated with worst survival
independently from regorafenib [125,169]. Nine micro-RNAs were associated with better overall
survival in patients receiving regorafenib [169]. Thus, in this trial, baseline prognostic factors associated
with survival were higher levels of AFP and c-MET, whereas predictive factors of better overall
survival in patients receiving regorafenib were ANG-1, cystatin-B, the latency-associated peptide of
transforming growth factor-beta 1 (LAPTGF ß1) and c-motif chemokine ligand 3 (MIP-1) [169]. Neither
AFP nor c-MET were predictive factors of regorafenib benefit and its efficacy was independent of
these biomarkers [125,169]. In another trial of sorafenib for prevention of HCC recurrence following
liver resection or RFA, none of the tested biomarkers related to angiogenesis and proliferation, gene
signatures, or mutations predicted sorafenib benefit over placebo [170].

4.3. Predictive Biomarkers Following Immunotherapy for HCC

Immunotherapy for cancer treatment has evolved into a complete novel paradigm. Cancer cells
avoid lymphocyte T cell activation and proliferation, highly expressing “programmed cell death
ligands” PD-1 ligand 1 and 2 (PD-L1, PD-L2) or “cytotoxic T lymphocyte protein 4” ligands (CTLA-4
and its ligands). The blockade of these unions through monoclonal antibodies (anti-PD-(L)1 or
Cells 2020, 9, 1370 14 of 27

anti-CTLA-4) unblocks the anti-cancer immune host response (“immune checkpoint inhibitors”).
The first anti-PD1 IgG4 monoclonal antibody available was nivolumab. Later, pembrolizumab (IgG4
anti-PD1), tremelimumab, and ipilimumab (IgG1 anti CTLA-4) and atezolizumab, durvalumab,
and avelumab (IgG1 anti-PD-L1) appeared.
Recently, the CheckMate-459 phase III, an open-label randomized clinical trial (RCT) assessed
nivolumab versus sorafenib in first-line therapy for patients with advanced HCC and did not show
a survival benefit of nivolumab over sorafenib [HR 0.85 (CI 0.72-1.02); P = 0.07]. Additionally, there was
not a significant difference in DFS, even showing a higher objective response rate (ORR) 15% vs. 7%.
Nevertheless, nivolumab showed lower rate of grade 3/4 adverse events (22% vs. 49%) (NCT02576509).
More recently, another phase III, open-label RCT, IMbrave-150 (NCT03434379) [171] showed
superiority of atezolizumab 1200 mg iv (anti-PD-L1) plus bevacizumab 15 mg/kg iv every 3 weeks
versus sorafenib [HR 0.58 (CI 0.42–0.79); P = 0.0006]. Eligibility criteria included preserved liver
function, systemic-naïve advanced HCC, ECOG 0-1 in the absence of main portal trunk invasion.
Longer progression-free survival (PFS) with significantly higher ORR (27% vs. 12%) and DCR of 74%
vs. 55% were observed. Similar incidence of all-grade adverse events and a lower incidence of grades
3/4 related adverse events were observed with the combination arm (36% vs. 46%). However, related
severe adverse events and treatment discontinuation were higher in atezolizumab + bevacizumab
arm (16% and 10%, respectively). Other combinations are being explored in phase III trials as first-line
treatments: pembrolizumab + lenvatinib vs. lenvatinib (LEAP-002, NCT03713593), sorafenib vs.
durvalumab (anti PDL-1) vs. durvalumab + tremelimumab (CTLA-4) (HIMALAYA, NCT03298451),
cabozantinib with/without atezolizumab versus sorafenib (COSMIC-312; NCT03755791) and nivolumab
+ ipilimumab versus sorafenib or lenvatinib (CheckMate-9DW; NCT04039607).
In second-line systemic treatment, for either intolerant patients or under tumor progression after
sorafenib, tremelimumab has been explored in an uncontrolled phase II trial in patients with HCV+
and was well tolerated [172]. Nivolumab was earlier explored in a phase I/II escalating and expansion
cohorts, uncontrolled trial, the CheckMate-040, which included Child–Pugh A–B < 9 patients under
progression with sorafenib (tolerant or intolerant) [3]. In this study, PD-L 1 expression on tumor
biopsies (≥1% vs. <1%) was not associated with better survival but higher ORRs were observed
(NCT02576509). Another phase III, double-blind RCT explored the treatment with pembrolizumab
200 mg iv every 3 weeks vs. placebo (KEYNOTE-240) in patients intolerant or under progression
with sorafenib, without main portal trunk invasion and with AFP levels <400 ng/mL [173]. The trial
was negative in terms of survival benefit according to its hypothesis test (13.9 vs. 10.6 months, HR of
0.78 (CI 0.71–0.99); P = 0.024). DFS did not reach its primary efficacy endpoint too. In a stratified
analysis, patients with AFP < 200 ng/mL or with post-progression (vs. intolerant to sorafenib) or with
HBV had the highest treatment benefit with pembrolizumab [173]. There is no robust data reporting
the effect of PD-L1 tumor expression as a predictive marker of response to pembrolizumab. Data on
PD-L1 tissue expression as a predictive tool with pembrolizumab was previously reported in a single
center uncontrolled intervention study in 29 patients with advanced HCC who had developed disease
progression or were intolerant to sorafenib [174]. In this study, serum pro and anti-inflammatory
cytokines, as well as serum levels of PD-1 and PD-L1 and PD-L2, were measured at baseline. In only
10 patients there was tumor tissue available and only 4 were positive to PD-L1 staining, showing higher
levels of plasma INF-y or IL-10 but PD-L1 staining was not correlated with plasma PD-L1 concentration.
Plasma tumor-growth factor β (TGF-β) levels were associated with overall survival [174].
Unfortunately, overall radiological responses in patients receiving immunotherapy ranged from
15% to 20% in studies testing single-agent immunotherapy either in first and second-line settings [3,173].
Additionally, PD-L1 expression on tumor tissue, although it was associated with higher radiological
responses, was not associated with a better survival benefit [3]. Thus, PD-L1 expression is not
mandatory to select candidates for the treatment with these agents. On the contrary, patients with
main portal trunk invasion or with high AFP values were excluded in these trials, as those with
Cells 2020, 9, 1370 15 of 27

autoimmune hepatitis or other immunological disorders, thus making it impossible to assess their role
in these scenarios.
Combining immunotherapy with anti-VEGF agents was explored to reduce VEGF-mediated
immunosuppression in HCC [175,176]. In the IMbrave study, the ORR increased to 27% with
a significant survival benefit over sorafenib [171]. However, until now, there are no predictive
biomarkers to better select the appropriate candidates for immunotherapy. Some exploratory analyses
were done in the phase 1b study of atezolizumab plus bevacizumab, showing that high expression of
PD-L1 in tumor tissue, higher expression of VEGF receptor 2, and higher T-regulatory cells immune
phenotype may be associated with better survival [177]. However, these biomarkers have not been
yet analyzed in the IMbrave trial [171]. In this study, in patients with AFP values above 400 ng/mL,
the combination arm did not show a significant benefit; still this should be cautiously analyzed [171].
A significant benefit was observed in patients with HBV related HCC [171].

5. Liquid Biopsy, Genomics and Other Biomarkers: The Future?

Finally, circulating free tumor deoxyribonucleic acid (ctDNA) in serum or plasma samples has
been the focus of novel researches. However, this approach called “liquid biopsy” has several issues to
be addressed [178–180]. Different forms of liquid biopsy have been described including circulating
tumor cells, ctDNA, microRNA, and extracellular vesicles. First, circulating tumor cells have been
assessed as predictors of treatment response in other tumors. However, the number of circulating
tumor cells is challenging, specifically in early stages. Second, ctDNA is another type of liquid biopsy.
However, it accounts for less than 1% of the total circulating cell-free DNA in normal conditions.
Additionally, ctDNA may not reflect specific tumor DNA, on the contrary it may reflect necrotic or
apoptotic tumor cells or cells that may not even originate from tumor tissue. Thus, the presence of
cancer-specific genetic or epigenetic DNA changes is a key factor to identify in these circumstances.
Moreover, these mutations maybe not specific for HCC in the context of cirrhosis, in which other DNA
mutations or bacterial DNA may circulate in peripheral blood. Nevertheless, liquid biopsy is being
researched not only for HCC early detection but also as a prognostic tool.
HCC is a heterogeneous tumor with substantial DNA mutations already reported. The most
frequent are those in TERT (the gene that encodes the catalytic subunit of telomerase), TP53 (the p53
tumor suppressor gene), and CTNNB1 (the β-Catenin gene) [80,81,181]. These mutations depend
on the underlying liver disease and are not completely present in most patients. TERT promoter
mutation is most frequently reported in 60% of HCC cases; whereas TP53 and CTNNB1 in around
30%. Sampling error of tissue biopsy or tumor heterogeneity may explain discrepancies observed in
circulating ctDNA mutations and those observed in tissue samples [180].
Genomic profiling of HCC has been developed during the last years. Two distinct classes have been
proposed based on the genomic profile and its correlation with phenotypic profiles. The proliferation
class has been associated with gene signatures of poor prognosis, TP53 mutations, and chromosomal
instability. This proliferation class has been linked to HBV infection, poor cell differentiation, higher
AFP values, and worse survival. On the contrary, the nonproliferation class has been associated
with CTNNB1 mutations, immune exclusion, HCV and alcoholic liver disease, low tumor grade,
lower frequency of vascular invasion, and better prognosis [80,81,182].
On the contrary, DNA methylation may be more specific. DNA methylation is an important
mechanism of epigenetic regulation of gene expression and has been reported in HCC [183].
Whether these specific tissue-specific methylation patterns, either on liquid biopsy or tumor samples,
are going to be useful biomarkers for early HCC diagnosis or prognosis is still uncertain and have not
yet been embraced in clinical practice.
Advancement of medical technology may further identify novel gene expression signatures or
new biomarkers such as those including ultimate sequencing identifying circulating or urine miRNAs,
genomic diversity [80,81,182], and epigenetic factors [183]. However, until now, no specific genomic
signature has been associated as a predictive marker of a better response to specific therapies for HCC.
Cells 2020, 9, 1370 16 of 27

Finally, radiomics in HCC is a novel but very preliminary approach based on artificial intelligence
and imaging data [184–186]. Imaging features are registered on machine-learning algorithms, and these
signs can assess and help HCC diagnosis and prognosis. However, radiomics is still a newborn
technology-based approach that needs internal and external validation.

6. Conclusions
HCC surveillance and early diagnosis demand a continuous search for improvement in clinical
tools for HCC screening due to its worldwide high associated mortality. The coexistence with
chronic liver disease and inflammation has counterbalanced the accuracy of several tumor biomarkers,
precluding them to be widely use in daily practice, except for AFP. Nevertheless, other tumor biomarkers
have been developed and have shown to be associated with poor prognosis in different HCC stages
and post-treatment assessment. Appropriate candidate selection for each therapeutic modality based
solely on these biomarkers is still far away from its clinical applicability in the clinical decision-making
processes. Ideal biomarkers for HCC are those that would enable clinicians to diagnose this cancer
at asymptomatic stages and also, to help and identify better candidates in each tumor stage for
appropriate therapeutic modalities [186,187]. So far, there is still a need for specific biomarkers to
improve detection of HCC at early or very early stages, assess specific prognosis and prediction of
treatment response.

Author Contributions: The manuscript was written by all co-authors. Specifically, M.D. wrote surveillance and
diagnosis; M.G.P. and F.P. wrote the HCC prognosis; F.P. wrote the treatment assessment. All authors have read
and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Conflicts of Interest: F Piñero has received Advisory Board and speaker honoraria and he is consultant for
RAFFO, BAYER Cono Sur and BAYER Andina; research grants from the Argentinean National Institute of Cancer
(INC ID-190), Argentinean National Ministry of Science and Technology Development (PICT 2017, FONCYT) and
from the Latin American Liver Research Educational and Awareness Network (LALREAN).

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